The present invention relates to DNA molecules responsible for encoding the multi-functional proteins that direct the biosynthesis of polyketide compounds, the products encoded by said DNA molecules, recombinant DNA expression vectors, and transformed microbial host cells.
Polyketides are a family of compounds that include a large number of structurally and functionally diverse natural products. For example, the polyketides provide the structural backbone for compounds that exhibit a variety of biological activities, such as, antibiotic, antitumor, and immunosuppressive agents.
Although the polyketides are quite diverse as indicated, they share a common mechanistic scheme of biosynthesis. The polyketides are synthesized by the successive condensation of small carboxylic acid residues followed by variable reduction steps at the resulting .beta.-keto carbon (i.e., .beta.-carbonyl) moiety in a process that is similar to the synthesis of fatty acids. The iterative synthetic process for many of these polyketides is controlled by a complex of large, multi-functional polypeptides that have distinct sites for the variety of activities that are required.
The general scheme for polyketide biosynthesis has been reviewed, for example, in Hopwood and Sherman, Annu. Rev. Genet., 1990, 24:37 and Katz and Donadio, Annu. Rev. Microbial., 1993, 47:875.
Naturally-occurring DNA sequences that encode the polyketide synthase enzymes have generally been found to be organized into repeated subunits, or modules, each of which encodes all the activities required in a single round of synthesis, which includes the condensation step itself and the post-condensation processing steps. Each activity is associated with a distinct site, which contributes to the specificity for the particular carboxylic acid building block that is incorporated at each condensation step, or which dictates the particular post-condensation processing functions that will be executed.
For example, PCT publication WO 93/13663 describes the organization of the gene encoding the polyketide synthase of Saccharopolyspora erythraea. The gene is organized in modules, with each module effecting one condensation step. The precise sequence of chain growth and the processing of the growing chain is determined by the genetic information in each module. This PCT application describes an approach for synthesizing novel polyketide structures by manipulating in several ways the DNA governing the biosynthesis of the cyclic lactone framework. In order to adapt this methodology to other polyketides, however, the DNA molecules directing the biosynthetic processing must first be isolated.
The present invention is directed to the DNA sequence for the gene cluster responsible for encoding tylactone synthase, the building machinery of tylactone, which is the basic building block of tylosin. As a result, the present invention enables modification of the DNA sequence so as to alter the type of carboxylic acids incorporated, the number of carboxylic acids incorporated, the post-condensation reactions performed, or any combination thereof, thereby resulting in novel tylosin-related polyketides.